The objective of the proposed research is to elucidate the mechanisms involved in the activation and regulation of the beta-glucoside utilization operon in E. coli K-12. The operon is silent in wold type cells but can be activated by a number of mutations. Once activated, the operon is induced in the presence of beta-glucosides. We wish to understand how DNA sequences upstream of the bgl regulatory region exert a negative effect on operon expression. We will use in vivo footprinting to identify and characterize the protein(s) involved. We will also analyze mutants which express the operon, with the aim of identifying functions which block expression. Operon expression is regulated by two proteins, one of which (BglF) acts as a negative regulator while the other (BglG) acts as a positive regulator. In the absence of inducer, the BglF protein, which also functions as the beta-glucoside transporter, phosphorylates BglG, thus blocking its action as a positive regulator. BglG acts as a transcriptional antiterminator, allowing transcripts to proceed through two rho-independent terminators which flank the first gene of the operon, bglG. We will elucidate the mechanism by which BglF phosphorylates BglG. Genetic approaches will be used to define sites of interaction action between the two proteins and the site of phosphorylation in BglG. The mechanism by which BglG effects transcriptional antitermination will be investigated. We will confirm that BglG is an RNA binding protein which recognizes specific sequences in bgl mRNA. The recognition sequence will be characterized by identifying mutations which eliminate recognition. An in vitro assay of antitermination will be developed to identify the factors that are involved in the antitermination process.